On Mar. 7th, 2022, Nature Cell Biology published a research article entitled “Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock” from Prof. Yujie Sun's group. Heat-shock transcription factor 1 (HSF1) orchestrates the fast and vast cellular response to heat shock (HS) through increased expression of heat shock proteins (HSPs). However, how HSF1 rapidly and reversibly regulates transcriptional reprogramming remains poorly defined. Combining super-resolution imaging, in vitro reconstitution, and high-throughput sequencing, researchers revealed an inducible and reversible phase-separation feedback mechanism for dynamic regulation of HSF1 activity to drive the transcriptional response and maintain protein homeostasis during acute stress.

　　As a conserved mechanism for various organisms to survive stress, the HS response is vital for maintaining protein homeostasis. HSF1 has been extensively studied due to its central role in regulating the HS response and restoring protein homeostasis. HSF1 orchestrates genome-wide transcriptional reprogramming to induce rapid and diverse changes in gene expression. In addition to acute stress, HSF1 also regulates a wide range of targets in various chronic processes and non-stress conditions such as cancers and neurodegenerative disease1. In contrast to malignant cancers, transcriptional activation during the HS response caused by acute stress is swift and vast, although HSF1 underlies the response in both cases. How HSF1 mechanistically promotes the remarkable transcriptional activation triggered by acute stress remains poorly understood. Decades of studies have established a comprehensive understanding of the activation and attenuation cycle of HSF1 during HS. However, the classic protein-DNA interaction-based model does not sufficiently explain the substantially different transcriptional responses caused by acute stress and in malignant cancers.

　　The role of liquid-liquid phase separation (LLPS) in transcription regulation has been reported extensively in recent years2. To explore whether LLPS plays a role in the regulation of HSF1, the researchers combined super-resolution imaging and in vitro reconstitution revealed that HSF1 forms condensates at both nSBs and HSP gene loci under HS. Furthermore, the PTMs identified within the LZ1-3 and RD upon HS have been characterized as positive or negative PTMs according to their effects on HSF1 target gene expression through alanine scan1,3,4. Therefore, they constructed a series of simulative mutants to investigate whether the opposite regulatory effects of different PTMs are the results of their effects on the LLPS of HSF1. The results showed that the phase separation capability of HSF1 is fine-tuned through PTMs at specific sites.

　　The research group next examined whether HSF1 LLPS in HS cells promote transcription of HSF1 target genes. Using dual-color STORM and Cut&Tag followed by high-throughput sequencing, they revealed that the LLPS capability of HSF1 is essential for the efficient recruitment of HSF1 and transcription apparatuses to HSP gene loci (Figure 1). Next, researchers investigated whether interfering with HSF1 LLPS affected the expression of its target genes using RNA-seq and qPCR. These results suggest that LLPS of HSF1 is the critical mechanism underlying the fast and vast transcriptional response under HS. Since HSF1 plays a role in tumorigenesis through changing either expression or PTMs, including Ser326 phosphorylation (S326P)5. However, the failure of HSF1 (S326D) to undergo LLPS indicated that transcriptional activation of HSF1 in malignant cells is not due to increased LLPS capability. They then revisited our RNA-seq results and found that cancer-specific HSF1 target genes were not activated in WT HS cells and LLPS-competent mutant M1 NHS cells. Therefore, these results demonstrated that HSF1 activates target expression via different mechanisms under HS conditions and in cancers.

　　Figure 1 HSF1 compartmentalizes transcription apparatuses.

　　Finally, researchers discovered that HSP70 disperses HSF1 condensates to attenuate transcription upon the cessation of HS and further prevents the gel-like phase-transition of HSF1 under extended HS stress.

　　The work revealed a phase-separation feedback mechanism for dynamic regulation of HSF1 activity to drive the transcriptional response and maintain protein homeostasis during acute stress (Figure 2). This inducible and reversible phase-separation feedback mechanism provides crucial, previously unknown information about how dynamic regulation of HSF1 activity drives transcriptional responses and attenuates HS to maintain protein homeostasis during acute stress.

　　Figure 2 Inducible and reversible LLPS of HSF1 mediates the transcriptional response during HS.

　　Ph.D. candidate Hongchen Zhang and Dr. Shipeng Shao are the co-first authors of the paper. Dr. Shipeng Shao and Prof. Yujie Sun are the co-corresponding authors of the paper. This work is supported by grants from the National Key R&D Program of China for Y.S. (No. 2017YFA0505300), the National Science Foundation of China (21825401 for Y.S., 31900898 for S.P., and 82070301 for Y.Z.), and the China Postdoctoral Science Foundation for S.P. (2019M660004 and 2019T120013).

　　Link: https://www.nature.com/articles/s41556-022-00846-7

　　References:

　　1 Gomez-Pastor, R., Burchfiel, E. T. & Thiele, D. J. Regulation of heat shock transcription factors and their roles in physiology and disease. Nat. Rev. Mol. Cell Biol. 19, 4-19, doi:10.1038/nrm.2017.73 (2018).

　　2 Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A Phase Separation Model for Transcriptional Control. Cell 169, 13-23, doi:10.1016/j.cell.2017.02.007 (2017).

　　3 Xu, Y. M., Huang, D. Y., Chiu, J. F. & Lau, A. T. Post-translational modification of human heat shock factors and their functions: a recent update by proteomic approach. J. Proteome Res. 11, 2625-2634, doi:10.1021/pr201151a (2012).

　　4 Guettouche, T., Boellmann, F., Lane, W. S. & Voellmy, R. Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress. BMC Biochem. 6, 4, doi:10.1186/1471-2091-6-4 (2005).

　　5 Mendillo, M. L. et al. HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell 150, 549-562, doi:10.1016/j.cell.2012.06.031 (2012).